Immobilized Metal-ion Affinity Chromatography Column Research Articles

Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns

Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals—Fe(II), Cu(II) and Ni(II)—on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.

The Activity and Cofactor Preferences of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Change Depending on Environmental Conditions

Actinomycetes, such as Mycobacterium species, are Gram-positive bacteria that utilize the small molecule mycothiol (MSH) as their primary reducing agent. Consequently, the enzymes involved in MSH biosynthesis are targets for drug development. The metal-dependent enzyme N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside to form 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside and acetate, the fourth overall step in MSH biosynthesis. Inhibitors of metalloenzymes typically contain a group that binds to the active site metal ion; thus, a comprehensive understanding of the native cofactor(s) of metalloenzymes is critical for the development of biologically effective inhibitors. Herein, we examined the effect of metal ions on the overall activity of MshB and probed the identity of the native cofactor. We found that the activity of MshB follows the trend Fe(2+) > Co(2+) > Zn(2+) > Mn(2+) and Ni(2+). Additionally, our results show that the identity of the cofactor bound to purified MshB is highly dependent on the purification conditions used (aerobic versus anaerobic), as well as the metal ion content of the medium during protein expression. MshB prefers Fe(2+) under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe(2+) and Zn(2+) under aerobic conditions as the metal content of the medium is altered. These results indicate that the cofactor bound to MshB under biological conditions is dependent on environmental conditions, suggesting that MshB may be a cambialistic metallohydrolase that contains a dynamic cofactor. Consequently, biologically effective inhibitors will likely need to dually target Fe(2+)-MshB and Zn(2+)-MshB.

Optimization of refolding with simultaneous purification of recombinant human granulocyte colony-stimulating factor from Escherichia coli by immobilized metal ion affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is a new technique for protein refolding, but it is limited to the refolding of fusion proteins with histidine affinity tags. In the present work, a non-fusion recombinant protein, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was successfully refolded with simultaneous purification by IMAC. rhG-CSF inclusion bodies solubilized in 8.0 mol/L urea was injected into a Cu(II)-iminodiacetic acid (IDA)-IMAC column, the soluble and active form of rhG-CSF in aqueous solution was obtained after desorbed from the column by linear increase of imidazole concentration. Several factors in the refolding process, including urea concentration and pH of mobile phases, type of buffer, glycerol concentration and loading sample volume, were investigated, respectively. When 200 μL of denatured/reduced rhG-CSF solution at a total protein concentration of 2.8 mg/mL was loaded on the IMAC column, rhG-CSF with a specific activity of 2.3 × 108 IU/mg and a mass recovery of 39% was obtained after IMAC refolding, and rhG-CSF was also purified during this chromatographic process, its purity was determined to be 97%.

Multipurpose peptide tags for protein isolation

A multifunctional peptide tag (HYDHYD) consisting of histidine, tyrosine and aspartate residues was fused to the N-terminal ends of green fluorescent protein (GFP), lactate dehydrogenase (LDH) and human hemoglobin (Hb), proteins which were subjected to ion-exchange chromatography (IEC), aqueous two-phase system partition, immobilized metal-ion affinity chromatography (IMAC), and hydrophobic interaction chromatography (HIC). Tagged GFP was retained significantly longer (>1 column volume) in both HIC and IEC. It exhibited 3× greater partition in favor of the hydrophobic phase in a two-phase system and 96% could be bound to an IMAC column which did not bind native GFP.

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.

Evaluation of immobilized metal-ion affinity chromatography for the fractionation of natural Cu complexing ligands

An immobilized metal-ion affinity chromatography (IMAC) method has been developed and validated for the separation of copper complexing ligands from soil solution. We first investigated the retention behavior of simple model ligands on the IMAC column and found that the ability to form ternary complexes of the structure Cu–IDA–ligand was the dominant factor influencing ligand retention on the IMAC column. The log K value of the Cu-complex was found to have only a minor influence on the retention. Legends containing only carboxylic acid functional groups were not retained on the column. To optimize reproducibility and quantitative recovery of copper ligands from soil solution, different composition and pH values of eluting buffer were tested. Soil solution chromatograms exhibited one non-retained fraction and two retained fractions. The elution times of the retained fractions were characteristic of peptides and proteins (first peak) and for compounds containing aromatic amines (second peak). The results show that IMAC is an effective tool for the fractionation of copper complexing ligands that are capable of forming ternary complexes.

Recovery and purification of aprotinin from industrial insulin-processing effluent by immobilized chymotrypsin and negative IMAC chromatographies

Aprotinin, a bovine protease inhibitor currently also produced in recombinant bacteria, yeast, and corn, has valuable applications as a human therapeutic and in tissue culture. The objective of this work was to develop the basis of a large-scale aprotinin purification process centered on immobilized metal ion affinity chromatography (IMAC). This technique uses ligands—metal ions—of a lower cost and higher stability than those traditionally used in affinity chromatography. Since aprotinin does not interact with IMAC ligands, collection is from the nonretained fractions (negative chromatography). Stirred-tank batch IMAC adsorption experiments indicated that one-step aprotinin purification could not be successful. Immobilized chymotrypsin chromatography was then used as a prepurification step, yielding a suitable feed for IMAC (with purification factors as high as 476). IMAC column fed with these prepurified materials produced purified aprotinin in the nonretained fractions with purification factors as high as 952.

Isolation and partial characterization of dissolved copper-complexing ligands in streamwaters.

We have separated two groups of copper-complexing ligands (the weak and strong ligands) from streamwaters in the Lake Biwa watershed by modified immobilized metal ion affinity chromatography (IMAC). The weak ligands were about 0.54-1.21% of the total dissolved organic matter (DOM), as determined by UV absorbance, and the strong ligands were about 0.06-0.21%. The results show that the stronger ligands were retained longer on the IMAC column, eluted later, and were accompanied by shorter wavelength UV absorbers, fluorescence maxima patterns with shorter wavelength excitation, and relatively "fresher" organic matter. The weak ligands with logK'(CuL) values of 6.6-7.7 had predominant humic-like fluorescence and may have been considerably degraded, while stronger ligands with logK'(CuL) values of 8.9-9.3 had only protein-like fluorescence and were relatively newly produced, labile material, as indicated from their amino acid composition. The protein-like fluorescence was mainly due to aromatic tryptophan probably bound to proteins or peptides. The results presented here have significant implications regarding the possible sources and biogeochemical role of organic ligands in aquatic environments.

Tandem Immobilized Metal-Ion Affinity Chromatography/Immunoaffinity Purification of His-tagged Proteins— Evaluation of Two Anti-His-Tag Monoclonal Antibodies

A tag comprising four to six histidines genetically fused to the protein of interest (His-tag) has been widely used to purify proteins by immobilized metal-ion affinity chromatography (IMAC). Here we report the utilization of the same tag twice in series, first for IMAC and subsequently for immunoaffinity purification. Both steps are based on completely different physical principles and can therefore remove different contaminants. Two anti-His-tag antibodies (3D5 and PentaHis) were characterized for their binding and elution properties using the BIAcore surface plasmon resonance biosensor. The dissociation constant of the PentaHis antibody was determined to be 1 × 10−8M and for the 3D5 antibody 3.4 × 10−7M at pH 7.4. Imidazole in the sample did interfere with binding, whereas chelating agents such as EDTA and high salt did not. The antibody 3D5 was coupled to a column matrix and used for a coupled two-step purification, in which the IMAC column is eluted with EDTA and the eluent is loaded directly on the immunoaffinity column. This method may constitute a very general procedure to purify proteins to near homogeneity without the need to tailor conditions individually, and it may thus be very attractive for high-throughput screening programs and for developing general protocols for clinical grade material.

Rapid, high-yield recovery of a recombinant digoxin binding single chain Fv from Escherichia coli.

We have isolated milligram quantities of active single chain antibody from the insoluble fraction of Escherichia coli cultures. The system relies on high-level expression from a T7 RNA polymerase-directed gene construct, 8 M urea to dissolve the desired protein out of the insoluble fraction, presumably inclusion bodies, isolation and concentration of the desired protein by nickel chelate [IDA-Ni(II)] immobilized metal-ion affinity chromatography (IMAC), and removal of urea from column fractions by dialysis directly into storage buffer. Routinely, about 50% of the protein loaded onto an IMAC column is recovered as single chain Fv at a concentration of approximately 0.7 mg/mL. As little as 3 days are required to obtain 10 mg of final product when starting with an overnight inoculum.

Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoproteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combination allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (∼0.7 μg) of bovine β-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.

Purification of synthetic peptides Immobilized metal ion affinity chromatography (IMAC)*

Immobilized metal ion affinity chromatography (IMAC) is a useful method for purification of synthetic peptides with an N-terminal metal-binding amino acid such as His, Trp, or Cys, especially when such residues are not present in other parts of the molecule. In solid phase peptide synthesis (SPPS), capping with acetic anhydride will, in principle, produce truncated peptides as the only side-products due to incomplete couplings. Consequently, only the desired product will carry the affinity label. Most of the impurities, therefore, can be removed by a single passage through an IMAC column. Some representative examples are presented, where fairly large peptides (30-40 amino acid residues) were efficiently purified by this approach.

Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins.

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.